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tactile sensilla on the ovipositor, tarsi and antennae of the flour moth, Ephestia kuehniella (Zeller) (Lepidoptera : Pyralidae)
Int. J. Insect Morphol. & Embryol., Vol. ~9, No. I, pp. 13-23, I99(I Printed in Great Britain
0020-7322/90 $3,00 + .1~) © 199{) Pergamon Press plc
S T R U C T U R E A N D DISTRIBUTION OF TACTILE A N D B I M O D A L TASTE/TACTILE SENSILLA ON THE OVIPOSITOR, TARSI A N D A N T E N N A E OF THE FLOUR MOTH, EPHESTIA K U E H N I E L L A (ZELLER) (LEPIDOPTERA • P Y R A L I D A E )
PETER ANDERSON* and ERIC HALLBERGt *Department of Ecology, Ecology Building, Helgonav~igen 5, S-223 62 Lund, Sweden; tDepartment of Zoology, Helgonav~igen 3, S-223 62 Lund. Sweden
(Accepted 26 October 1989)
Abstract--The bimodal taste/tactile sensilla on ovipositor, tarsi and antennae and the tactile sensilla on ovipositor and tarsi of the flour moth, Ephestia kuehniella (Zeller) (Lepidoptera : Pyralidae), were studied with electron microscopy to provide a structural base for further physiological and behavioural studies. These sensilla can be involved in the perception of the oviposition-deterring pheromone secreted by the larvae, as well as for the general assessment of the oviposition site. The taste/tactile sensilla in the 3 different positions have slightly divergent morphological characteristics; on the antennae and tarsi, there are 4 chemosensory neurons, while on the ovipositor there are 5. There is also a mechano-sensory unit connected to the base of the bristle of these sensilla. Tactile sensilla, innervated by one sensory cell, are present on the tarsi and on 2 different positions of the ovipositor. Freeze-substitution technique for transmission electron microscopy was employed on the tarsi and yielded superior results compared with conventional chemical fixation. Index descriptors (in addition to those in title): ODP, SEM, TEM, freeze-substitution. INTRODUCTION WHEN l a r v a e o f the M e d i t e r r a n e a n flour m o t h , Ephestia kuehniella, a p e s t o f s t o r e d p r o d u c t s , m e e t h e a d to h e a d , t h e y d e p o s i t d r o p l e t s o f p h e r o m o n e on t h e s u b s t r a t u m , which o r i g i n a t e f r o m t h e i r m a n d i b u l a r g l a n d s ( C o r b e t , 1971). O v i p o s i t i n g Ephestia f e m a l e s r e s p o n d to this s e c r e t i o n b y r e l a t i n g the n u m b e r o f eggs t h e y lay, to t h e a m o u n t o f p h e r o m o n e at t h e o v i p o s i t i o n site. L o w c o n c e n t r a t i o n s o f t h e p h e r o m o n e a r e slightly o v i p o s i t i o n - s t i m u l a t i n g , w h e r e a s high c o n c e n t r a t i o n s a r e s t r o n g l y d e t e r r i n g ( C o r b e t , 1973). T h e s e c r e t i o n also f u n c t i o n s as a k a i r o m o n e for the f e m a l e s o f t h e p a r a s i t i c w a s p Venturia canescens ( G r a y . ) ( H y m e n o p t e r a : I c h n e u m o n i d a e ) , which use the s e c r e t i o n to l o c a t e h o s t l a r v a e ( C o r b e t , 1971). F r o m t h e larval m a n d i b u l a r g l a n d s e c r e t i o n o f E. kuehniella, 16 d i f f e r e n t , s t r u c t u r a l l y r e l a t e d , [~-ketones h a v e b e e n i d e n t i f i e d ( M u d d , 1978, 1981, 1983). T h e s e s u b s t a n c e s h a v e b e e n s h o w n to s t i m u l a t e t h e o v i p o s i t i o n in V. canescens ( M u d d a n d C o r b e t , 1982). H o w e v e r , t h e i r i n f l u e n c e o n o v i p o s i t i n g Ephestia-females has n o t y e t b e e n s t u d i e d . 13
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P. ANDERSONand E. HALLBERG
C o n t a c t c h e m o s e n s i l l a ( t a s t e / t a c t i l e sensilla) p r e s e n t on tarsi a n d / o r o v i p o s i t o r in insects, h a v e b e e n s h o w n to b e i n v o l v e d in t h e c h o i c e of f o o d - p l a n t , o v i p o s i t i o n site a n d t r a n s f e r o f i n f o r m a t i o n b e t w e e n c o n s p e c i f i c i n d i v i d u a l s (St/idler, 1984). In m o s t s t u d i e d cases in insects, t h e o v i p o s i t i o n - d e t e r r i n g p h e r o m o n e s ( O D P s ) h a v e b e e n f o u n d to b e l o w - v o l a t i l e ( P r o k o p y , 1981) a n d c o n s e q u e n t l y c o n t a c t c h e m o r e c e p t o r s s h o u l d b e i n v o l v e d in t h e p e r c e p t i o n of t h e p h e r o m o n e ( P r o k o p y , 1972; P r o k o p y a n d S p a t c h e r , 1977). W e d e s c r i b e h e r e t h e m o r p h o l o g i c a l u l t r a s t r u c t u r e of the t a s t e / t a c t i l e sensilla on o v i p o s i t o r , tarsi, a n d a n t e n n a e o f E. kuehnieUa, as a basis for a p r o j e c t w h e r e the b e h a v i o u r a l r e s p o n s e s o f f e m a l e s to t h e larval m a n d i b u l a r g l a n d s e c r e t i o n will b e i n v e s t i g a t e d . U p to n o w , t h e r e is no i n f o r m a t i o n a v a i l a b l e on the s e n s o r y basis for the p e r c e p t i o n o f O D P in t h e f e m a l e s of this species. A l s o , t h e tactile bristles a r e d e s c r i b e d since t h e y o c c u r t o g e t h e r o n tarsi a n d o v i p o s i t o r , a n d h a v e a similar e x t e r n a l s h a p e . F u r t h e r , d i f f e r e n t m e t h o d s o f tissue p r o c e s s i n g ( c o n v e n t i o n a l c h e m i c a l fixation a n d f r e e z e - s u b s t i t u t i o n ) w e r e e m p l o y e d to s t u d y a n d e v a l u a t e t h e tissue p r e s e r v a t i o n of tarsi, o v i p o s i t o r , a n d a n t e n n a e o f E. kuehniella. T h e tarsi are e x t r e m e l y difficult to fix p r o p e r l y with c h e m i c a l f i x a t i o n , o w i n g to t h e small d i a m e t e r , p o s s e s s i o n o f scales a n d t h e g e n e r a l h y d r o p h o b i c i t y o f the cuticle. F r e e z e - s u b s t i t u t i o n was e m p l o y e d , since this m e t h o d e a r l i e r has s h o w n to give b e t t e r p r e s e r v a t i o n of similar tissue in insects ( S t e i n b r e c h t , 1985).
MATERIALS
AND METHODS
Insects
The moths were obtained from a laboratory culture, which was reared on wheat-bran. The culture was maintained at 26°C, 40% RH and at a 12 : 12 LD regime.
Fixation For transmission electron microscopy (TEM), the specimens were either fixed in a conventional fixative (tarsi, ovipositor and antenna) or freeze-substituted (tarsi). For conventional fixation, the ovipositor was first exposed by applying a slight pressure on the abdomen and then the fixative (ice-cold 4% glutaraldehyde in 0.2 M cacodylate buffer) was injected. After a few min, the ovipositor was excised and placed in the abovementioned fixative over-night. The specimens were washed in buffer and post-fixed in 1% osmium tetroxide for 2 hr. The specimens were further washed in buffer, dehydrated in ethanol and finally embedded in Epon. For freeze-substitution, the tarsi were plunge-frozen (entry velocity 2 m/s) into liquified propane, transferred into osmium tetroxide/acetone at dry-ice temperature and slowly warmed to room temperature after 3 days (Steinbrecht, 1985). The specimens were thereafter embedded in Epon and polymerized at 60°C. For TEM, ultra-thin sections were cut with a diamond knife on a LKB ultrotome. The stained sections (LKB ultrostainer) were examined in a Jeol 1200 EX or a Zeiss EM 10 transmission electron microscope. For scanning electron microscopy (SEM), the specimens were fixed in glutaraldehyde as for TEM, or in 70% ethanol. The tarsi were sonicated before this procedure to remove the scales. The specimens were dehydrated in ethanol, transferred to Freon TF and critical-point dried. The mounted specimens were sputtered in a Polaron E5400 high resolution sputter coater and finally studied in a Jeol T 330 scanning electron microscope.
RESULTS
Fixation techniques T h e s p e c i m e n s , w h i c h w h e r e f i x e d with c o n v e n t i o n a l f i x a t i o n , e x h i b i t e d a n u m b e r o f f e a t u r e s , c o n n e c t e d w i t h a s u b - o p t i m a l f i x a t i o n , such as swollen m i t o c h o n d r i a , i r r e g u l a r m e m b r a n e s a n d a g e n e r a l low c o n t r a s t . A s a c o n t r a s t , c r y o - s u b s t i t u t e d s p e c i m e n s o f t h e
Structure and distributionof sensilla of Ephestia kuehniella
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tarsi showed well-preserved tissues, specially towards the periphery, whereas there were some traces of crystallization in the central parts of the tarsi. The membranes appeared smooth and continuous and were easy to trace. The mitochondria had the typical appearance of freeze-substituted ones, with a moderately dark matrix, and very electrondense cristae. The shapes of the mitochondria are somewhat irregular. The depth of the properly preserved tissue was about 10 Ixm, when freeze-substitution was applied. This depth usually includes a considerable portion of the sensillum, with its sensory and enveloping cells. Structure and distribution of sensilla The sensilla of the ovipositor and tarsi belong to 2 structural/functional main types: tactile sensilla and bimodal taste/tactile sensilla. The structural characteristics of these sensilla are somewhat divergent depending on their position. Structure of sensilla on the ovipositor. The ovipositor is just over 1 mm long: in cross section its circumference is round, except in the distal pointed portion that is dorsoventrally flattened, being provided with a ventral furrow. Hair-like sensilla are present at 2 different positions; proximally about the middle part, and distally at the above-mentioned flattened part (Fig. 1). Tactile sensilla are present in both positions, whereas taste/tactile sensilla only are present at the distal position. The distal tactile sensilla have a pointed termination and the length of the bristle varies between 30 and 200 i~m (Fig. 2). The longer hairs have a basal diameter of about 3 txm, the shorter about 1.5 Ixm. In the proximal tactile sensilla, the basal diameter of the bristle is 2-3 I~m and their length is 100-200 I~m. The hair-shaft has longitudinal ridges and is evenly tapering towards the distal part that forms a pointed tip. In the tactile sensiUa the sockets tightly surround the base of the hair-shafts, in contrast to those of the taste/tactile sensilla (Fig. 3). There is also a difference between the proximal and distal tactile sensilla, since the former have more narrow and flatter sockets. The lumen of the hair-shaft is filled with a spongy extracellular material. The hair base is inserted into the surrounding cuticle and the terminal part of the sensory cell (sensory process) is embedded in the basal part of the hair. The sensory cell terminates as an electron dense tubular body, which is cone-shaped, having a pointed outline in longitudinal section. Below the pointed termination, the diameter of the tubular body increases; at the same time, the outline of the sensory process becomes quite irregular in transverse section. However, the shape is bilaterally symmetric being essentially bow-shaped, with the convex side facing the cuticle. Beneath the socket, the sensory process is surrounded by an electron dense sheath, which completely encloses the sensory process. An outer sensillum lymph cavity surrounds the sensory process. The bimodal taste/tactile sensilla can be distinguished in the SEM by the shape of the hair and the socket structures (Figs 2, 3). The hairs are comparatively stouter than the tactile hairs and their sockets are wider and lower. The difference in shape between taste/ tactile and tactile sensilla can also be seen in an ordinary light microscope. In contrast to the tactile hairs, the hair-base (when viewed in the SEM) is not closely fitting into the socket itself, but is separated by 0.5 I~m and connected via a membrane (Fig. 3). The variation in length of these sensilla is also less than in the tactile sensilla; 30-60 i~m. The basal diameter is about 3 I~m. These hairs have a terminal pore, and the hollow liquid-filled interior of the hair shaft
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FIG. 1. Lateral view of ovipositor of Ephestia kuehniella. Distal sensilla are found between arrows. A r r o w h e a d s point to proximal sensilla. SEM, scale bar = 100 I~m. FIG. 2. Sensilla of distal part of the ovipositor belong to 2 types: taste/tactile type (TT), which have a blunt tip, and tactile type (mechanoreceptive) (MR), which end in a pointed tip, Ventral side of ovipositor is to the right. SEM; scale bar = 10 [xm. FIG. 3. Sockets of tactile sensilla (MR) tightly surround base of hair-shaft, while sockets of taste/ tactile (qT) sensilla are wider. SEM; scale bar = 5 txm. FIG. 4. Transverse section through a taste/tactile bristle showing 5 chemosensory units (arrowheads). T E M ; scale bar = 0.5 ~tm.
Structure and distributionof sensilla of Ephestia Kuehniella
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houses 5 unbranched sensory processes, which reach this pore. The sensory processes are located within a separate cuticular tube, which is adhering to the inner surface of the shaft (Fig, 4). The cuticle of the hair-shaft is provided with a pore-tubule-like system being penetrated by an arrangement of electron dense filaments. This pore-tubule-like system is built up by tubules that are connected with both the surface and the lumen of the hair. The tubules that have diameter of about 20 nm, form a convoluted tubular system about 0.2 ixm below the outer surface of the hair, which has a total thickness of 0.4-0.5 I~m in the middle and basal parts. At the inner surface, the tubules are connected with electron-dense filaments, with a diameter of about 20 nm, that are suspended in the hair lumen. In the basal parts below the hair, the 5 long sensory processes are enclosed in a sheath originating from the innermost enveloping cell. The arrangement of the enveloping cells conforms with the common, i.e. in addition to the innermost (thecogen) cell, there are 2 outer enveloping cells (trichogen and tormogen cells). This sheath is continuous with the cuticular tube mentioned above. At the level of the junction hair/surrounding cuticle, there is a 6th sensory process, terminating with a tubular body, thus indicative of a mechanoreceptive function. This tubular body is flattened, measuring 0.4 x 0.8 I~m in its basal region. Distribution ofsensilla on the ovipositor. Three types of sensilla are found: taste/tactile sensilla, long (75-180 I~m) and short (<50 p,m) tactile sensilla (Fig. 1). On the most distal, flattened segment of the ovipositor, about 100 sensiila are present. Equal numbers (35-40) of long and short tactile sensilla are evenly scattered over the ventral side of the segment. About 10 taste/tactile sensilla are found ventrally, predominantly on the distal part of the segment. Laterally, 30 long tactile sensilla are present. On the middle part of the ovipositor, about 50 long tactile sensilla are scattered evenly around the circumference of the ovipositor. Structure ofsensilla on the tarsi. The same types of sensilla are present on the tarsi as on the ovipositor. However, there are some variations: the tactile sensilla have usually shorter and stouter shafts (20 ~xm long and 2.7 ixm wide). As in the ovipositor sensilla, these are innervated by one sensory celT. Also, the bimodal taste/tactile sensilla are shorter and stouter on the tarsi, being only 15-30 Ixm long, and have a basal diameter of 2-2.5 Ixm. They are innervated by 4 chemoreceptive cells and one mechanoreceptive (Fig. 7). The pore-tubule-like system is similar to that of the sensilla of the ovipositor, but appears better preserved, probably owing to the freeze-substitution techniques. In the basal part of the hair, the filaments (diameter 20 nm) that adhere to the inner surface of the hair form an electron dense meshwork in the outer receptor cavity (Fig. 8). There, they are suspended without any evident connection to the enveloping or sensory cells. The tubular body has in cross section semicircular outline, measuring 0.3 x 0.6 Ixm. Distribution o f sensilla on the tarsi. There is no pronounced difference in the distribution of bimodal tactile/taste sensilla on the tarsi between males and females. Neither could any difference in the distribution of sensilla between the different pairs of legs (fore-, mid- and hindlegs) be found, except for the 1st tarsomer. On the 2nd, 3rd and 4th tarsomers, 2 ventral and 2 lateral sensilla, one on each side, are usually present (Fig. 5). The 5th tarsomer has 4 lateral sensilla, but a varying number of ventral sensilla. Two sensilla are always present on the distal edge, and then an additional 1-6 sensilla appear
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FI~. 5. A schematic view of arrangement of sensilla on ventral surface of the 4th and 5th tarsomer of a fore-leg. Arrows indicate tactile sensilla, arrowheads taste/tactile sensilla classified as laterally directed, while ventrally directed taste/tactile sensilla are unmarked. Scale bar = 20 ~.m. on the mid and proximal part of the 5th tarsomer (Figs 5, 6). On the 1st tarsomer no ventral sensiUa are found. The pattern of lateral sensilla is not consistent between the pairs of legs. The forelegs have 6 lateral sensilla, while the mid- and hindlegs usually have only 0-2 lateral sensilla, although exceptionally up to 5 can be found. One long taste/ tactile sensillum occurs ventrally between the pulvilli (Figs 5, 6). Dorsally, taste/tactile sensiUa occur occasionally on all tarsomers. When present, usually only one per tarsomer, except on the 1st tarsomer when 2--4 sensilla can be found. Only 2 tactile sensilla occur on the tarsi. They are positioned ventrally on the distal edge of the 5th tarsomere (Figs 5, 6). Structure o f sensilla on the antennae. Taste/tactile sensilla (sensilla chaetica) are present also on the antennae. They have a bristle, which is about 20-30 ixm long and a basal diameter of about 2 I~m. These sensilla are easily recognizable owing to the socket structures (Fig. 10). In contrast to the taste/tactile sensilla found on tarsi and ovipositor, those present on the antennae have transversely arranged ridges on the bristle. The taste sensilla on the antennae are innervated by 4 chemosensory cells and one mechanosensory cell, in a pattern similar to that of the corresponding type on the tarsi (Fig. 11). The tubular body has the same shape and size, as in the taste/tactile sensilla of the tarsi. The surface pattern consists mainly of transversely arranged ridges (Fig. 10). Tactile sensilta, of the type found on the ovipositor and the tarsi, are not present on the antennae. Distribution o f sensilla on the antennae. Usually there are 3 taste/tactile sensilla on each antennal segment: one on the ventral side is displaced towards the distal part of the segment, and 2 laterally, close to the scales that cover the dorsal side (Fig. 9). These latter sensilla are located approximately on the middle part of the segment.
DISCUSSION Freeze-fixation of invertebrate sensilla, followed by freeze-substitution, has been employed on insect and to a lesser extent crustacean tissues (Steinbrecht, 1980; Haug and Altner, 1984). Especially, antennae and their olfactory sensilla have been studied using these methods. They generally give a superior tissue preservation and are thus
Structure and distribution of sensilla of Ephestia kuehniella
FI6. 6. Distal part of 5th tarsomer has a number of taste/tactile (arrowheads), and tactile (arrows) sensilla. SEM; scale bar = 20 ixm. FIG. 7. In basal part of hair of taste/tactile sensillum of tarsi, there are 4 chemosensory units (arrows) and one mechanosensory, which are developed as a tubular body (TB). In lymph cavity (LC), there are electron-dense filaments (F) that originate from inside of hair lumen. TEM; scale bar = 0.5 i~m. Fie. 8. Below hair, processes of sensory cells (arrowheads;, are present in a lymph cavity (LC) and are surrounded by enveloping cells (EC). TEM; scale bar = 0.5 I~m.
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FIG. 9. Taste/tactile sensilla (arrows) on antennae SEM; scale bar = 10 l~m. FIG. 10. Taste/tactile sensilla on antennae have transversely arranged cuticular ridges (arrows). SEM; scale bar = 2 p.m. F~G. 11. A transverse section through socket of an antennal taste/tactile sensillum with one mechanosensory (TB) and 4 chemosensory(arrowheads) units. TEM; scale bar = 0.5 ~m.
especially suitable for morphometric and cytoarchitectural studies of cells and tissues (Keil, 1984; Steinbrecht and Gnatzy, 1984). Freeze-fixation and freeze-substitution employed on the tarsi of Ephestia yielded properly fixed and preserved tissue specimens, both to the tactile and the bimodal taste/tactile sensilla and to the surrounding tissues. The scales that are present on the tarsi, as well as the general hydrophobity of the cuticle render the underlying tissue less exposed to the ordinary fixative fluids, and therefore the low-temperature methods are promising in this type of tissue. Especially, the penetration of osmium tetroxide appears to be impaired by the barrier formed by the cuticle; here, the treatment, including a prolonged infiltration with osmium tetroxide in acetone, probably enhances the availability of osmium, which results in an increased contrast. The
Structure and distribution of sensilla of Ephestia kuehniella
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small diameter of the tarsi is also favouring when considering the chances for a good tissue preservation using these techniques. A common characteristic of the sensory organs on tarsi and ovipositor in Lepidoptera, is the presence of 2 distinct types of hair-like sensilla; one tactile and one bimodal taste/ tactile sensillum. Bimodal taste/tactile sensilla are also found on the antennae of Ephestia, whereas hair-like tactile sensilla are lacking on the antennae. The bimodal taste/tactile sensilla (contact chemosensilla) are of an ubiquitous type that is found in most insect groups. They have been identified on all major parts of the insect body, including the ovipositor, the antennae and the wings. Most frequently, they occur on the mouthparts and the tarsi (St~idler, 1984). The pore-tubule-like system in the taste/tactile sensilla of E. kuehniella, has also been described in other species (Gaffal, 1979). Silver particles can penetrate the hair shaft, and it has been proposed that the pore-tubule-like system provides a pathway for small molecules (Gaffal, 1979). It remains to be demonstrated, if this system is active in the stimulus conducting processes, or if the stimulus only can reach the sensory cells through the apical pore of the hair. In general, contact chemosensilla of this type are innervated by 5 receptor cells, 4 of which are chemoreceptive and one is a mechanoreceptor (Zacharuk, 1985). Each receptor cell is tuned to a certain class of compounds, to which it gives the strongest response (St~idler, 1984). Contact chemoreceptors have been shown to be involved in the perception of the chemical environment of the female. Information about host plant, oviposition site, con- and interspecifics can be perceived by contact chemoreceptors in insects (St~idler, 1984). The structure and distribution of the ovipositor sensilla has been investigated in a number of Lepidopteran species (Chadha and Roome, 1980; Faucheux and Chauvin, 1981). In the studied species, there are 2 basic types of sensilla: tactile sensilla with a hair of variable length and bimodal taste/tactile sensilla, that are present towards the distal part of the ovipositor. It is interesting to note that the bimodal sensilla on the ovipositor in Ephestia, as well as in Spodoptera littoralis (Chadha and Roome, 1980) have 5 chemoreceptive cells, while on the antennae and tarsi, the corresponding type has 4 chemoreceptive cells. In Chilo partellus there are either 4 or 5 chemoreceptive cells (Chadna and Roome, 1980). The usual number of chemoreceptive cells in this sensillar type is 4 (Zacharuk, 1985), since the receptor cells are tuned to one class of compounds, the possession of an additional receptor in the sensilla on the ovipositor can indicate a wider perception range (Stoffolano and ¥in, 1987). In C. partellus the sensilla on the ovipositor are suggested to prevent the female from ovipositing on chemically unacceptable surfaces (Chadha and Roome, 1980). Studies of Pieris brassicae (L.) (Lepidoptera : Pieridae) indicate that contact chemosensilla on the ovipositor are involved in the detection of the ODP of this species (Klijnstra, 1982). Tarsal contact chemoreceptors have also been shown to be involved in the perception of chemical substances active between conspecifics (pheromones). Both sexual and deterring pheromones have been found to be perceived in this way (St~idler, 1984). In P. brassicae tarsal contact chemosensilla are sensitive to salts, water, host-plant compounds and to the ODP (Ma and Schoonhoven, 1973; Klijnstra, 1982). Also, the antennal contact chemoreceptors may be involved in these types of responses. Most identified ODPs so far, have been shown to be low-volatile and water-soluble (Prokopy, 1981), which suggests short range perception. Although the ODP of E. kuehniella is an exception, since the compounds identified from the larval mandibular glands are not
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P. ANDERSON and E. HALLBERG
soluble in water (Mudd, 1978, 1981, 1983), the low volatility of these suggests that they are perceived by contact chemoreception. In the fruit flies, Rhagoletis pomonella (Walch) and R. cerasi (L.) (Diptera : Tephritidae), behavioural tests with ovipositing females, suggested that direct contact with the O D P - m a r k e d fruit was necessary for the detection of the O D P (Prokopy, 1972, Katsoyannos, 1975). T h e oviposition behaviour of E. kuehniella is in some aspects similar to that of R. pomonella. In both species, the female walks over the oviposition substrate, while searching for a suitable oviposition site. During the search, she gets in contact with the possible O D P - m a r k i n g on the oviposition substrate and adjusts her oviposition behaviour to her findings. Since larvae of E. kuehniella secrete a p h e r o m o n e to the substrate f r o m their mandibular glands when they meet, the amount of p h e r o m o n e at a site is correlated to the population density. To prevent overcrowding and extensive competition for food for her offspring, the female of E. kuehniella can adjust the n u m b e r of eggs she lays at an oviposition site (Corbet, 1973). In R. pomonella chemosensilla sensitive to O D P , were located to the distal ventrolateral portions of the 2nd, 3rd and 4th tarsomers on all legs (Prokopy and Spatcher, 1977; Crnjar and Prokopy, 1982). On the tarsi or E. kuehniella we found contact chemosensilla located ventrally and laterally in positions suitable for perception of the O D P . No sexual dimorphism was found in the n u m b e r or arrangement of the sensilla, which could have indicated specific ODP-sensitive receptors on the female. Hopefully, behavioural and electrophysiological tests will show whether the tarsal sensiUa are involved in p h e r o m o n e perception. Thus, the same tactile and taste/tactile types are found on the tarsi and ovipositor of Ephestia and the total sensory function, i.e. contact chemoreception and a tactile sense of the sensilla in these 2 different locations can be similar. As indicated above, the sensilla in these 2 positions are involved in oviposition, but they are active during different stages of the oviposition sequence. The tarsal sensilla are most probably m o r e frequently used than those of the ovipositor that are exposed less frequently in a specialized behavioural situation: during oviposition and possibly also when calling. In the latter case, however, the sensilla are not brought in contact with the substrate, which could activate them. Finally, this mapping of the taste/tactile and tactile sensilla of E. kuehniella demonstrates the similarity with earlier studied ODP-receptive systems. Our further studies will hopefully show if and how the sensilla are involved in the perception of the O D P produced by the larvae in E. kuehniella.
Acknowledgements--We are grateful to Prof. Jan L6fqvist for encouraging this project and for valuable advice. We are also grateful for all the help, both with the cryo-substitution methods and the manuscript, which has been provided by Professor R. A. Steinbrecht (Max-Planck-Institutfur Verhaltensphysiologie, Seewiesen, Federal Republic of Germany). Lastly, we also thank our colleagues at the Department of Animal Ecology for their comments on the manuscript. The study was supported by grants from the Swedish Natural Science Research Council (Grant 8998-100 to E. Hallberg), and from the Swedish Council for Forestry and Agricultural Research (Grant 333/88 J312 : 2 to Prof. Jan L6fqvist).
REFERENCES CHADHA, G. K. and R. E. ROOME. 1980. Oviposition behaviour and the sensiUa of Chilo partellus and Spodoptera littoralis (Lepidoptera : Noctuidae). J. Zool. Lond. 192: 169-78. CORRET,S. A. 1971. Mandibular gland secretion of larvae of the flour moth, Anagasta kuehniella, contains an epideitic pheromone and elicits oviposition movements in a Hymenopteran parasite. Nature (Lond.) 232: 481-84.
Structure and distribution of sensilla of Ephestia kuehniella
23
CoraEt, S. A. 1973. Oviposition pheromone in larval mandibular glands of Ephestia kuehniella. Nature (Lond.) 243: 537-38. CRNJAR, R. M. and R. J. PROKOPY. 1982. Morphological and electrophysiological mapping of tarsal chemoreceptors of oviposition-deterring pheromone in Rhagoletis pomoneUa flies. J. Insect Physiol. 28: 393-400. FAUCHEUX, M. J. and G. CHAUVlN. 1981. Les r6cepteurs sensorieels impliqu6s dans la recherche du support de ponte chez Tinea pellionella L. (Lepidoptera : Tineidae). Arch. Zool. Exp. G~n. 122: 5-16. GAFFAL, K, P. 1979. An ultrastructural study of the tips of four classical bimodal sensilla with one mechanosensitive and several chemo-sensitive receptor cells. Zoomorphologie 92: 273-91. HAUG, T. and H. ALINER. 1984. A cryo-fixation study of a subcuticular receptor organ in the antennular tip of the terrestrial isopod Porcellio scaber Latr. (Crustacea). J. Ultrastruct. Res. 87: 62-74. KATSOYANNOS, B. I. 1975. Oviposition-deterring, male-arresting, fruit-marking pheromone in Rhagoletis cerasi. Env. EntomoL 4: 801-07. KFLL, T. A, 1984. Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (lnsecta. Lepidoptera). Zoomorphology 104: 147-56. KLIJNSTRA,J. W. 1982. Perception of the oviposition deterrent pheromone in Pieris brassicae, pp. 145-151, ht J. H. VlssEr and A. K. MINKS (eds) Insect-Plant Relationships. Pudoc, Wagenhingen. MA, W. C. and L. M. SCHOONHOVEN. 1973. Tarsal contact cbemosensory hairs of the large white butterfly Pieris brassicae and their possible role in oviposition behaviour. Entomol. Exp. Appl. 16: 343-57. MUDD, A. 1978. Novel B-triketones from Lepidoptera. J. Chem. Soc. Chem. Commun. 1978: 1075-76. MUDD, A. 1981. Novel 2-acylcyclohexane-l,3-diones in the mandibular glands of lepidopteran larvae. Kairomones of Ephestia kuehniella Zeller. J. Chem. Soc. Perkin Trans. 1981: 2357-62. MIJDD, A. 1983. Further novel 2-acylcyclohexane-l,3-diones from lepidopteran larvae. J. Chem. Soc. Perkin Trans. 1983: 2161-64. MUDD, A. and S. A. CORBEt. 1982. Response of the Ichneumonid parasite Nemeritis canescens to kairomones from the flour moth, Ephestia kuehniella. J. Chem. Ecol. 8: 843-5{). PROKOPY, r . J. 1972. Evidence for a marking pheromone deterring repeated oviposition in apple maggot flies. Env. Entomol. 4: 298-300. PROKOPY, R. J. 1981. Epideitic pheromones that influence spacing patterns of phytophagous insects, pp. 428432. In D. A. NORDLUND, R. L. JONES and W. J. LEwis (eds) Semiochemicals: Their Role in Pest Control. J. Wiley & Sons, New York. PROKOPY, R. J. and P. J. SPATCHER 1977. Location of receptors for oviposition-deterring pheromone in Rhagoletis pomonella flies. Annu. Rev. EntomoL 70: 96(P62. SIEINBRECHt, R. A. 1980. Cryofixation without cryoprotectants. Freeze-substitution and freeze-etching of an insect olfactory receptor. Tissue Cell 12: 73-100. STEINBRECHT, R. A. 1985. Recrystallization and ice-crystal growth in a biological specimen, as shown by a simple freeze substitution method. J. Microsc. 140: 41-46. STEINBRECHT, R. A, and W. GNATZY. 1984. Pheromone receptors in Bombyx mori and Anteraea pernyi: 1. Reconstruction of the cellular organization of the sensilla trichodea. Cell Tissue Res. 235: 25-34. STOFFOLANO,Jr J. G. and L. R. S, YIN. 1987. Structure and function of the ovipositor and associated sensilla of the apple maggot, Rhagoletis pomonella (Walch)(Diptera : Tephritidae), Int. J. Insect Morphol. Embryol. 16: 41-69. ST,~DLER,E. 1984. Contact chemoreception, pp. 3-35. In W. J. BELL and R. T. CARDI~(eds) Chemical Ecology' of Insects. Chapman & Hall, London. ZACHARUK, R. Y. 1985. Antennae and sensilla, pp. 1-69. In G. A. KErKOt and L. I. GILBERT (eds) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 6. Pergamon Press, Oxford.
0020-7322/90 $3,00 + .1~) © 199{) Pergamon Press plc
S T R U C T U R E A N D DISTRIBUTION OF TACTILE A N D B I M O D A L TASTE/TACTILE SENSILLA ON THE OVIPOSITOR, TARSI A N D A N T E N N A E OF THE FLOUR MOTH, EPHESTIA K U E H N I E L L A (ZELLER) (LEPIDOPTERA • P Y R A L I D A E )
PETER ANDERSON* and ERIC HALLBERGt *Department of Ecology, Ecology Building, Helgonav~igen 5, S-223 62 Lund, Sweden; tDepartment of Zoology, Helgonav~igen 3, S-223 62 Lund. Sweden
(Accepted 26 October 1989)
Abstract--The bimodal taste/tactile sensilla on ovipositor, tarsi and antennae and the tactile sensilla on ovipositor and tarsi of the flour moth, Ephestia kuehniella (Zeller) (Lepidoptera : Pyralidae), were studied with electron microscopy to provide a structural base for further physiological and behavioural studies. These sensilla can be involved in the perception of the oviposition-deterring pheromone secreted by the larvae, as well as for the general assessment of the oviposition site. The taste/tactile sensilla in the 3 different positions have slightly divergent morphological characteristics; on the antennae and tarsi, there are 4 chemosensory neurons, while on the ovipositor there are 5. There is also a mechano-sensory unit connected to the base of the bristle of these sensilla. Tactile sensilla, innervated by one sensory cell, are present on the tarsi and on 2 different positions of the ovipositor. Freeze-substitution technique for transmission electron microscopy was employed on the tarsi and yielded superior results compared with conventional chemical fixation. Index descriptors (in addition to those in title): ODP, SEM, TEM, freeze-substitution. INTRODUCTION WHEN l a r v a e o f the M e d i t e r r a n e a n flour m o t h , Ephestia kuehniella, a p e s t o f s t o r e d p r o d u c t s , m e e t h e a d to h e a d , t h e y d e p o s i t d r o p l e t s o f p h e r o m o n e on t h e s u b s t r a t u m , which o r i g i n a t e f r o m t h e i r m a n d i b u l a r g l a n d s ( C o r b e t , 1971). O v i p o s i t i n g Ephestia f e m a l e s r e s p o n d to this s e c r e t i o n b y r e l a t i n g the n u m b e r o f eggs t h e y lay, to t h e a m o u n t o f p h e r o m o n e at t h e o v i p o s i t i o n site. L o w c o n c e n t r a t i o n s o f t h e p h e r o m o n e a r e slightly o v i p o s i t i o n - s t i m u l a t i n g , w h e r e a s high c o n c e n t r a t i o n s a r e s t r o n g l y d e t e r r i n g ( C o r b e t , 1973). T h e s e c r e t i o n also f u n c t i o n s as a k a i r o m o n e for the f e m a l e s o f t h e p a r a s i t i c w a s p Venturia canescens ( G r a y . ) ( H y m e n o p t e r a : I c h n e u m o n i d a e ) , which use the s e c r e t i o n to l o c a t e h o s t l a r v a e ( C o r b e t , 1971). F r o m t h e larval m a n d i b u l a r g l a n d s e c r e t i o n o f E. kuehniella, 16 d i f f e r e n t , s t r u c t u r a l l y r e l a t e d , [~-ketones h a v e b e e n i d e n t i f i e d ( M u d d , 1978, 1981, 1983). T h e s e s u b s t a n c e s h a v e b e e n s h o w n to s t i m u l a t e t h e o v i p o s i t i o n in V. canescens ( M u d d a n d C o r b e t , 1982). H o w e v e r , t h e i r i n f l u e n c e o n o v i p o s i t i n g Ephestia-females has n o t y e t b e e n s t u d i e d . 13
14
P. ANDERSONand E. HALLBERG
C o n t a c t c h e m o s e n s i l l a ( t a s t e / t a c t i l e sensilla) p r e s e n t on tarsi a n d / o r o v i p o s i t o r in insects, h a v e b e e n s h o w n to b e i n v o l v e d in t h e c h o i c e of f o o d - p l a n t , o v i p o s i t i o n site a n d t r a n s f e r o f i n f o r m a t i o n b e t w e e n c o n s p e c i f i c i n d i v i d u a l s (St/idler, 1984). In m o s t s t u d i e d cases in insects, t h e o v i p o s i t i o n - d e t e r r i n g p h e r o m o n e s ( O D P s ) h a v e b e e n f o u n d to b e l o w - v o l a t i l e ( P r o k o p y , 1981) a n d c o n s e q u e n t l y c o n t a c t c h e m o r e c e p t o r s s h o u l d b e i n v o l v e d in t h e p e r c e p t i o n of t h e p h e r o m o n e ( P r o k o p y , 1972; P r o k o p y a n d S p a t c h e r , 1977). W e d e s c r i b e h e r e t h e m o r p h o l o g i c a l u l t r a s t r u c t u r e of the t a s t e / t a c t i l e sensilla on o v i p o s i t o r , tarsi, a n d a n t e n n a e o f E. kuehnieUa, as a basis for a p r o j e c t w h e r e the b e h a v i o u r a l r e s p o n s e s o f f e m a l e s to t h e larval m a n d i b u l a r g l a n d s e c r e t i o n will b e i n v e s t i g a t e d . U p to n o w , t h e r e is no i n f o r m a t i o n a v a i l a b l e on the s e n s o r y basis for the p e r c e p t i o n o f O D P in t h e f e m a l e s of this species. A l s o , t h e tactile bristles a r e d e s c r i b e d since t h e y o c c u r t o g e t h e r o n tarsi a n d o v i p o s i t o r , a n d h a v e a similar e x t e r n a l s h a p e . F u r t h e r , d i f f e r e n t m e t h o d s o f tissue p r o c e s s i n g ( c o n v e n t i o n a l c h e m i c a l fixation a n d f r e e z e - s u b s t i t u t i o n ) w e r e e m p l o y e d to s t u d y a n d e v a l u a t e t h e tissue p r e s e r v a t i o n of tarsi, o v i p o s i t o r , a n d a n t e n n a e o f E. kuehniella. T h e tarsi are e x t r e m e l y difficult to fix p r o p e r l y with c h e m i c a l f i x a t i o n , o w i n g to t h e small d i a m e t e r , p o s s e s s i o n o f scales a n d t h e g e n e r a l h y d r o p h o b i c i t y o f the cuticle. F r e e z e - s u b s t i t u t i o n was e m p l o y e d , since this m e t h o d e a r l i e r has s h o w n to give b e t t e r p r e s e r v a t i o n of similar tissue in insects ( S t e i n b r e c h t , 1985).
MATERIALS
AND METHODS
Insects
The moths were obtained from a laboratory culture, which was reared on wheat-bran. The culture was maintained at 26°C, 40% RH and at a 12 : 12 LD regime.
Fixation For transmission electron microscopy (TEM), the specimens were either fixed in a conventional fixative (tarsi, ovipositor and antenna) or freeze-substituted (tarsi). For conventional fixation, the ovipositor was first exposed by applying a slight pressure on the abdomen and then the fixative (ice-cold 4% glutaraldehyde in 0.2 M cacodylate buffer) was injected. After a few min, the ovipositor was excised and placed in the abovementioned fixative over-night. The specimens were washed in buffer and post-fixed in 1% osmium tetroxide for 2 hr. The specimens were further washed in buffer, dehydrated in ethanol and finally embedded in Epon. For freeze-substitution, the tarsi were plunge-frozen (entry velocity 2 m/s) into liquified propane, transferred into osmium tetroxide/acetone at dry-ice temperature and slowly warmed to room temperature after 3 days (Steinbrecht, 1985). The specimens were thereafter embedded in Epon and polymerized at 60°C. For TEM, ultra-thin sections were cut with a diamond knife on a LKB ultrotome. The stained sections (LKB ultrostainer) were examined in a Jeol 1200 EX or a Zeiss EM 10 transmission electron microscope. For scanning electron microscopy (SEM), the specimens were fixed in glutaraldehyde as for TEM, or in 70% ethanol. The tarsi were sonicated before this procedure to remove the scales. The specimens were dehydrated in ethanol, transferred to Freon TF and critical-point dried. The mounted specimens were sputtered in a Polaron E5400 high resolution sputter coater and finally studied in a Jeol T 330 scanning electron microscope.
RESULTS
Fixation techniques T h e s p e c i m e n s , w h i c h w h e r e f i x e d with c o n v e n t i o n a l f i x a t i o n , e x h i b i t e d a n u m b e r o f f e a t u r e s , c o n n e c t e d w i t h a s u b - o p t i m a l f i x a t i o n , such as swollen m i t o c h o n d r i a , i r r e g u l a r m e m b r a n e s a n d a g e n e r a l low c o n t r a s t . A s a c o n t r a s t , c r y o - s u b s t i t u t e d s p e c i m e n s o f t h e
Structure and distributionof sensilla of Ephestia kuehniella
15
tarsi showed well-preserved tissues, specially towards the periphery, whereas there were some traces of crystallization in the central parts of the tarsi. The membranes appeared smooth and continuous and were easy to trace. The mitochondria had the typical appearance of freeze-substituted ones, with a moderately dark matrix, and very electrondense cristae. The shapes of the mitochondria are somewhat irregular. The depth of the properly preserved tissue was about 10 Ixm, when freeze-substitution was applied. This depth usually includes a considerable portion of the sensillum, with its sensory and enveloping cells. Structure and distribution of sensilla The sensilla of the ovipositor and tarsi belong to 2 structural/functional main types: tactile sensilla and bimodal taste/tactile sensilla. The structural characteristics of these sensilla are somewhat divergent depending on their position. Structure of sensilla on the ovipositor. The ovipositor is just over 1 mm long: in cross section its circumference is round, except in the distal pointed portion that is dorsoventrally flattened, being provided with a ventral furrow. Hair-like sensilla are present at 2 different positions; proximally about the middle part, and distally at the above-mentioned flattened part (Fig. 1). Tactile sensilla are present in both positions, whereas taste/tactile sensilla only are present at the distal position. The distal tactile sensilla have a pointed termination and the length of the bristle varies between 30 and 200 i~m (Fig. 2). The longer hairs have a basal diameter of about 3 txm, the shorter about 1.5 Ixm. In the proximal tactile sensilla, the basal diameter of the bristle is 2-3 I~m and their length is 100-200 I~m. The hair-shaft has longitudinal ridges and is evenly tapering towards the distal part that forms a pointed tip. In the tactile sensiUa the sockets tightly surround the base of the hair-shafts, in contrast to those of the taste/tactile sensilla (Fig. 3). There is also a difference between the proximal and distal tactile sensilla, since the former have more narrow and flatter sockets. The lumen of the hair-shaft is filled with a spongy extracellular material. The hair base is inserted into the surrounding cuticle and the terminal part of the sensory cell (sensory process) is embedded in the basal part of the hair. The sensory cell terminates as an electron dense tubular body, which is cone-shaped, having a pointed outline in longitudinal section. Below the pointed termination, the diameter of the tubular body increases; at the same time, the outline of the sensory process becomes quite irregular in transverse section. However, the shape is bilaterally symmetric being essentially bow-shaped, with the convex side facing the cuticle. Beneath the socket, the sensory process is surrounded by an electron dense sheath, which completely encloses the sensory process. An outer sensillum lymph cavity surrounds the sensory process. The bimodal taste/tactile sensilla can be distinguished in the SEM by the shape of the hair and the socket structures (Figs 2, 3). The hairs are comparatively stouter than the tactile hairs and their sockets are wider and lower. The difference in shape between taste/ tactile and tactile sensilla can also be seen in an ordinary light microscope. In contrast to the tactile hairs, the hair-base (when viewed in the SEM) is not closely fitting into the socket itself, but is separated by 0.5 I~m and connected via a membrane (Fig. 3). The variation in length of these sensilla is also less than in the tactile sensilla; 30-60 i~m. The basal diameter is about 3 I~m. These hairs have a terminal pore, and the hollow liquid-filled interior of the hair shaft
16
P. ANDERSON and E. HALLBERG
FIG. 1. Lateral view of ovipositor of Ephestia kuehniella. Distal sensilla are found between arrows. A r r o w h e a d s point to proximal sensilla. SEM, scale bar = 100 I~m. FIG. 2. Sensilla of distal part of the ovipositor belong to 2 types: taste/tactile type (TT), which have a blunt tip, and tactile type (mechanoreceptive) (MR), which end in a pointed tip, Ventral side of ovipositor is to the right. SEM; scale bar = 10 [xm. FIG. 3. Sockets of tactile sensilla (MR) tightly surround base of hair-shaft, while sockets of taste/ tactile (qT) sensilla are wider. SEM; scale bar = 5 txm. FIG. 4. Transverse section through a taste/tactile bristle showing 5 chemosensory units (arrowheads). T E M ; scale bar = 0.5 ~tm.
Structure and distributionof sensilla of Ephestia Kuehniella
17
houses 5 unbranched sensory processes, which reach this pore. The sensory processes are located within a separate cuticular tube, which is adhering to the inner surface of the shaft (Fig, 4). The cuticle of the hair-shaft is provided with a pore-tubule-like system being penetrated by an arrangement of electron dense filaments. This pore-tubule-like system is built up by tubules that are connected with both the surface and the lumen of the hair. The tubules that have diameter of about 20 nm, form a convoluted tubular system about 0.2 ixm below the outer surface of the hair, which has a total thickness of 0.4-0.5 I~m in the middle and basal parts. At the inner surface, the tubules are connected with electron-dense filaments, with a diameter of about 20 nm, that are suspended in the hair lumen. In the basal parts below the hair, the 5 long sensory processes are enclosed in a sheath originating from the innermost enveloping cell. The arrangement of the enveloping cells conforms with the common, i.e. in addition to the innermost (thecogen) cell, there are 2 outer enveloping cells (trichogen and tormogen cells). This sheath is continuous with the cuticular tube mentioned above. At the level of the junction hair/surrounding cuticle, there is a 6th sensory process, terminating with a tubular body, thus indicative of a mechanoreceptive function. This tubular body is flattened, measuring 0.4 x 0.8 I~m in its basal region. Distribution ofsensilla on the ovipositor. Three types of sensilla are found: taste/tactile sensilla, long (75-180 I~m) and short (<50 p,m) tactile sensilla (Fig. 1). On the most distal, flattened segment of the ovipositor, about 100 sensiila are present. Equal numbers (35-40) of long and short tactile sensilla are evenly scattered over the ventral side of the segment. About 10 taste/tactile sensilla are found ventrally, predominantly on the distal part of the segment. Laterally, 30 long tactile sensilla are present. On the middle part of the ovipositor, about 50 long tactile sensilla are scattered evenly around the circumference of the ovipositor. Structure ofsensilla on the tarsi. The same types of sensilla are present on the tarsi as on the ovipositor. However, there are some variations: the tactile sensilla have usually shorter and stouter shafts (20 ~xm long and 2.7 ixm wide). As in the ovipositor sensilla, these are innervated by one sensory celT. Also, the bimodal taste/tactile sensilla are shorter and stouter on the tarsi, being only 15-30 Ixm long, and have a basal diameter of 2-2.5 Ixm. They are innervated by 4 chemoreceptive cells and one mechanoreceptive (Fig. 7). The pore-tubule-like system is similar to that of the sensilla of the ovipositor, but appears better preserved, probably owing to the freeze-substitution techniques. In the basal part of the hair, the filaments (diameter 20 nm) that adhere to the inner surface of the hair form an electron dense meshwork in the outer receptor cavity (Fig. 8). There, they are suspended without any evident connection to the enveloping or sensory cells. The tubular body has in cross section semicircular outline, measuring 0.3 x 0.6 Ixm. Distribution o f sensilla on the tarsi. There is no pronounced difference in the distribution of bimodal tactile/taste sensilla on the tarsi between males and females. Neither could any difference in the distribution of sensilla between the different pairs of legs (fore-, mid- and hindlegs) be found, except for the 1st tarsomer. On the 2nd, 3rd and 4th tarsomers, 2 ventral and 2 lateral sensilla, one on each side, are usually present (Fig. 5). The 5th tarsomer has 4 lateral sensilla, but a varying number of ventral sensilla. Two sensilla are always present on the distal edge, and then an additional 1-6 sensilla appear
18
P. ANDERSONand E. HALLBERG
FI~. 5. A schematic view of arrangement of sensilla on ventral surface of the 4th and 5th tarsomer of a fore-leg. Arrows indicate tactile sensilla, arrowheads taste/tactile sensilla classified as laterally directed, while ventrally directed taste/tactile sensilla are unmarked. Scale bar = 20 ~.m. on the mid and proximal part of the 5th tarsomer (Figs 5, 6). On the 1st tarsomer no ventral sensiUa are found. The pattern of lateral sensilla is not consistent between the pairs of legs. The forelegs have 6 lateral sensilla, while the mid- and hindlegs usually have only 0-2 lateral sensilla, although exceptionally up to 5 can be found. One long taste/ tactile sensillum occurs ventrally between the pulvilli (Figs 5, 6). Dorsally, taste/tactile sensiUa occur occasionally on all tarsomers. When present, usually only one per tarsomer, except on the 1st tarsomer when 2--4 sensilla can be found. Only 2 tactile sensilla occur on the tarsi. They are positioned ventrally on the distal edge of the 5th tarsomere (Figs 5, 6). Structure o f sensilla on the antennae. Taste/tactile sensilla (sensilla chaetica) are present also on the antennae. They have a bristle, which is about 20-30 ixm long and a basal diameter of about 2 I~m. These sensilla are easily recognizable owing to the socket structures (Fig. 10). In contrast to the taste/tactile sensilla found on tarsi and ovipositor, those present on the antennae have transversely arranged ridges on the bristle. The taste sensilla on the antennae are innervated by 4 chemosensory cells and one mechanosensory cell, in a pattern similar to that of the corresponding type on the tarsi (Fig. 11). The tubular body has the same shape and size, as in the taste/tactile sensilla of the tarsi. The surface pattern consists mainly of transversely arranged ridges (Fig. 10). Tactile sensilta, of the type found on the ovipositor and the tarsi, are not present on the antennae. Distribution o f sensilla on the antennae. Usually there are 3 taste/tactile sensilla on each antennal segment: one on the ventral side is displaced towards the distal part of the segment, and 2 laterally, close to the scales that cover the dorsal side (Fig. 9). These latter sensilla are located approximately on the middle part of the segment.
DISCUSSION Freeze-fixation of invertebrate sensilla, followed by freeze-substitution, has been employed on insect and to a lesser extent crustacean tissues (Steinbrecht, 1980; Haug and Altner, 1984). Especially, antennae and their olfactory sensilla have been studied using these methods. They generally give a superior tissue preservation and are thus
Structure and distribution of sensilla of Ephestia kuehniella
FI6. 6. Distal part of 5th tarsomer has a number of taste/tactile (arrowheads), and tactile (arrows) sensilla. SEM; scale bar = 20 ixm. FIG. 7. In basal part of hair of taste/tactile sensillum of tarsi, there are 4 chemosensory units (arrows) and one mechanosensory, which are developed as a tubular body (TB). In lymph cavity (LC), there are electron-dense filaments (F) that originate from inside of hair lumen. TEM; scale bar = 0.5 i~m. Fie. 8. Below hair, processes of sensory cells (arrowheads;, are present in a lymph cavity (LC) and are surrounded by enveloping cells (EC). TEM; scale bar = 0.5 I~m.
19
20
P. ANDERSONand E. HALLBERG
FIG. 9. Taste/tactile sensilla (arrows) on antennae SEM; scale bar = 10 l~m. FIG. 10. Taste/tactile sensilla on antennae have transversely arranged cuticular ridges (arrows). SEM; scale bar = 2 p.m. F~G. 11. A transverse section through socket of an antennal taste/tactile sensillum with one mechanosensory (TB) and 4 chemosensory(arrowheads) units. TEM; scale bar = 0.5 ~m.
especially suitable for morphometric and cytoarchitectural studies of cells and tissues (Keil, 1984; Steinbrecht and Gnatzy, 1984). Freeze-fixation and freeze-substitution employed on the tarsi of Ephestia yielded properly fixed and preserved tissue specimens, both to the tactile and the bimodal taste/tactile sensilla and to the surrounding tissues. The scales that are present on the tarsi, as well as the general hydrophobity of the cuticle render the underlying tissue less exposed to the ordinary fixative fluids, and therefore the low-temperature methods are promising in this type of tissue. Especially, the penetration of osmium tetroxide appears to be impaired by the barrier formed by the cuticle; here, the treatment, including a prolonged infiltration with osmium tetroxide in acetone, probably enhances the availability of osmium, which results in an increased contrast. The
Structure and distribution of sensilla of Ephestia kuehniella
21
small diameter of the tarsi is also favouring when considering the chances for a good tissue preservation using these techniques. A common characteristic of the sensory organs on tarsi and ovipositor in Lepidoptera, is the presence of 2 distinct types of hair-like sensilla; one tactile and one bimodal taste/ tactile sensillum. Bimodal taste/tactile sensilla are also found on the antennae of Ephestia, whereas hair-like tactile sensilla are lacking on the antennae. The bimodal taste/tactile sensilla (contact chemosensilla) are of an ubiquitous type that is found in most insect groups. They have been identified on all major parts of the insect body, including the ovipositor, the antennae and the wings. Most frequently, they occur on the mouthparts and the tarsi (St~idler, 1984). The pore-tubule-like system in the taste/tactile sensilla of E. kuehniella, has also been described in other species (Gaffal, 1979). Silver particles can penetrate the hair shaft, and it has been proposed that the pore-tubule-like system provides a pathway for small molecules (Gaffal, 1979). It remains to be demonstrated, if this system is active in the stimulus conducting processes, or if the stimulus only can reach the sensory cells through the apical pore of the hair. In general, contact chemosensilla of this type are innervated by 5 receptor cells, 4 of which are chemoreceptive and one is a mechanoreceptor (Zacharuk, 1985). Each receptor cell is tuned to a certain class of compounds, to which it gives the strongest response (St~idler, 1984). Contact chemoreceptors have been shown to be involved in the perception of the chemical environment of the female. Information about host plant, oviposition site, con- and interspecifics can be perceived by contact chemoreceptors in insects (St~idler, 1984). The structure and distribution of the ovipositor sensilla has been investigated in a number of Lepidopteran species (Chadha and Roome, 1980; Faucheux and Chauvin, 1981). In the studied species, there are 2 basic types of sensilla: tactile sensilla with a hair of variable length and bimodal taste/tactile sensilla, that are present towards the distal part of the ovipositor. It is interesting to note that the bimodal sensilla on the ovipositor in Ephestia, as well as in Spodoptera littoralis (Chadha and Roome, 1980) have 5 chemoreceptive cells, while on the antennae and tarsi, the corresponding type has 4 chemoreceptive cells. In Chilo partellus there are either 4 or 5 chemoreceptive cells (Chadna and Roome, 1980). The usual number of chemoreceptive cells in this sensillar type is 4 (Zacharuk, 1985), since the receptor cells are tuned to one class of compounds, the possession of an additional receptor in the sensilla on the ovipositor can indicate a wider perception range (Stoffolano and ¥in, 1987). In C. partellus the sensilla on the ovipositor are suggested to prevent the female from ovipositing on chemically unacceptable surfaces (Chadha and Roome, 1980). Studies of Pieris brassicae (L.) (Lepidoptera : Pieridae) indicate that contact chemosensilla on the ovipositor are involved in the detection of the ODP of this species (Klijnstra, 1982). Tarsal contact chemoreceptors have also been shown to be involved in the perception of chemical substances active between conspecifics (pheromones). Both sexual and deterring pheromones have been found to be perceived in this way (St~idler, 1984). In P. brassicae tarsal contact chemosensilla are sensitive to salts, water, host-plant compounds and to the ODP (Ma and Schoonhoven, 1973; Klijnstra, 1982). Also, the antennal contact chemoreceptors may be involved in these types of responses. Most identified ODPs so far, have been shown to be low-volatile and water-soluble (Prokopy, 1981), which suggests short range perception. Although the ODP of E. kuehniella is an exception, since the compounds identified from the larval mandibular glands are not
22
P. ANDERSON and E. HALLBERG
soluble in water (Mudd, 1978, 1981, 1983), the low volatility of these suggests that they are perceived by contact chemoreception. In the fruit flies, Rhagoletis pomonella (Walch) and R. cerasi (L.) (Diptera : Tephritidae), behavioural tests with ovipositing females, suggested that direct contact with the O D P - m a r k e d fruit was necessary for the detection of the O D P (Prokopy, 1972, Katsoyannos, 1975). T h e oviposition behaviour of E. kuehniella is in some aspects similar to that of R. pomonella. In both species, the female walks over the oviposition substrate, while searching for a suitable oviposition site. During the search, she gets in contact with the possible O D P - m a r k i n g on the oviposition substrate and adjusts her oviposition behaviour to her findings. Since larvae of E. kuehniella secrete a p h e r o m o n e to the substrate f r o m their mandibular glands when they meet, the amount of p h e r o m o n e at a site is correlated to the population density. To prevent overcrowding and extensive competition for food for her offspring, the female of E. kuehniella can adjust the n u m b e r of eggs she lays at an oviposition site (Corbet, 1973). In R. pomonella chemosensilla sensitive to O D P , were located to the distal ventrolateral portions of the 2nd, 3rd and 4th tarsomers on all legs (Prokopy and Spatcher, 1977; Crnjar and Prokopy, 1982). On the tarsi or E. kuehniella we found contact chemosensilla located ventrally and laterally in positions suitable for perception of the O D P . No sexual dimorphism was found in the n u m b e r or arrangement of the sensilla, which could have indicated specific ODP-sensitive receptors on the female. Hopefully, behavioural and electrophysiological tests will show whether the tarsal sensiUa are involved in p h e r o m o n e perception. Thus, the same tactile and taste/tactile types are found on the tarsi and ovipositor of Ephestia and the total sensory function, i.e. contact chemoreception and a tactile sense of the sensilla in these 2 different locations can be similar. As indicated above, the sensilla in these 2 positions are involved in oviposition, but they are active during different stages of the oviposition sequence. The tarsal sensilla are most probably m o r e frequently used than those of the ovipositor that are exposed less frequently in a specialized behavioural situation: during oviposition and possibly also when calling. In the latter case, however, the sensilla are not brought in contact with the substrate, which could activate them. Finally, this mapping of the taste/tactile and tactile sensilla of E. kuehniella demonstrates the similarity with earlier studied ODP-receptive systems. Our further studies will hopefully show if and how the sensilla are involved in the perception of the O D P produced by the larvae in E. kuehniella.
Acknowledgements--We are grateful to Prof. Jan L6fqvist for encouraging this project and for valuable advice. We are also grateful for all the help, both with the cryo-substitution methods and the manuscript, which has been provided by Professor R. A. Steinbrecht (Max-Planck-Institutfur Verhaltensphysiologie, Seewiesen, Federal Republic of Germany). Lastly, we also thank our colleagues at the Department of Animal Ecology for their comments on the manuscript. The study was supported by grants from the Swedish Natural Science Research Council (Grant 8998-100 to E. Hallberg), and from the Swedish Council for Forestry and Agricultural Research (Grant 333/88 J312 : 2 to Prof. Jan L6fqvist).
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23
CoraEt, S. A. 1973. Oviposition pheromone in larval mandibular glands of Ephestia kuehniella. Nature (Lond.) 243: 537-38. CRNJAR, R. M. and R. J. PROKOPY. 1982. Morphological and electrophysiological mapping of tarsal chemoreceptors of oviposition-deterring pheromone in Rhagoletis pomoneUa flies. J. Insect Physiol. 28: 393-400. FAUCHEUX, M. J. and G. CHAUVlN. 1981. Les r6cepteurs sensorieels impliqu6s dans la recherche du support de ponte chez Tinea pellionella L. (Lepidoptera : Tineidae). Arch. Zool. Exp. G~n. 122: 5-16. GAFFAL, K, P. 1979. An ultrastructural study of the tips of four classical bimodal sensilla with one mechanosensitive and several chemo-sensitive receptor cells. Zoomorphologie 92: 273-91. HAUG, T. and H. ALINER. 1984. A cryo-fixation study of a subcuticular receptor organ in the antennular tip of the terrestrial isopod Porcellio scaber Latr. (Crustacea). J. Ultrastruct. Res. 87: 62-74. KATSOYANNOS, B. I. 1975. Oviposition-deterring, male-arresting, fruit-marking pheromone in Rhagoletis cerasi. Env. EntomoL 4: 801-07. KFLL, T. A, 1984. Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (lnsecta. Lepidoptera). Zoomorphology 104: 147-56. KLIJNSTRA,J. W. 1982. Perception of the oviposition deterrent pheromone in Pieris brassicae, pp. 145-151, ht J. H. VlssEr and A. K. MINKS (eds) Insect-Plant Relationships. Pudoc, Wagenhingen. MA, W. C. and L. M. SCHOONHOVEN. 1973. Tarsal contact cbemosensory hairs of the large white butterfly Pieris brassicae and their possible role in oviposition behaviour. Entomol. Exp. Appl. 16: 343-57. MUDD, A. 1978. Novel B-triketones from Lepidoptera. J. Chem. Soc. Chem. Commun. 1978: 1075-76. MUDD, A. 1981. Novel 2-acylcyclohexane-l,3-diones in the mandibular glands of lepidopteran larvae. Kairomones of Ephestia kuehniella Zeller. J. Chem. Soc. Perkin Trans. 1981: 2357-62. MIJDD, A. 1983. Further novel 2-acylcyclohexane-l,3-diones from lepidopteran larvae. J. Chem. Soc. Perkin Trans. 1983: 2161-64. MUDD, A. and S. A. CORBEt. 1982. Response of the Ichneumonid parasite Nemeritis canescens to kairomones from the flour moth, Ephestia kuehniella. J. Chem. Ecol. 8: 843-5{). PROKOPY, r . J. 1972. Evidence for a marking pheromone deterring repeated oviposition in apple maggot flies. Env. Entomol. 4: 298-300. PROKOPY, R. J. 1981. Epideitic pheromones that influence spacing patterns of phytophagous insects, pp. 428432. In D. A. NORDLUND, R. L. JONES and W. J. LEwis (eds) Semiochemicals: Their Role in Pest Control. J. Wiley & Sons, New York. PROKOPY, R. J. and P. J. SPATCHER 1977. Location of receptors for oviposition-deterring pheromone in Rhagoletis pomonella flies. Annu. Rev. EntomoL 70: 96(P62. SIEINBRECHt, R. A. 1980. Cryofixation without cryoprotectants. Freeze-substitution and freeze-etching of an insect olfactory receptor. Tissue Cell 12: 73-100. STEINBRECHT, R. A. 1985. Recrystallization and ice-crystal growth in a biological specimen, as shown by a simple freeze substitution method. J. Microsc. 140: 41-46. STEINBRECHT, R. A, and W. GNATZY. 1984. Pheromone receptors in Bombyx mori and Anteraea pernyi: 1. Reconstruction of the cellular organization of the sensilla trichodea. Cell Tissue Res. 235: 25-34. STOFFOLANO,Jr J. G. and L. R. S, YIN. 1987. Structure and function of the ovipositor and associated sensilla of the apple maggot, Rhagoletis pomonella (Walch)(Diptera : Tephritidae), Int. J. Insect Morphol. Embryol. 16: 41-69. ST,~DLER,E. 1984. Contact chemoreception, pp. 3-35. In W. J. BELL and R. T. CARDI~(eds) Chemical Ecology' of Insects. Chapman & Hall, London. ZACHARUK, R. Y. 1985. Antennae and sensilla, pp. 1-69. In G. A. KErKOt and L. I. GILBERT (eds) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 6. Pergamon Press, Oxford.